Keywords. Phytoalexins; mycorrhizae; cowpea; Glomus fasciculatum; disease control.

Transcription

1 J. Biosci., Vol. 18, Number 2, June 1993, pp Printed in India. Induction and accumulation of phytoalexins in cowpea roots infected with a mycorrhizal fungus Glomus fasciculatum and their resistance to Fusarium wilt disease Ρ SUNDARESAN, Ν UBALTHOOSE RAJA and Ρ GUNASEKARAN Department of Microbiology, School of Biological Sciences, Madurai Kamaraj University, Madurai , India MS received 10 January 1992 Abstract. The interaction of a vesicular-arbuscular mycorrhizal fungus Glomus fasciculatum with a wilt-causing soil borne pathogen, Fusarium oxysporum, was studied in cowpea (Vigna unguiculata). It was found that pre-establishment by vesicular-arbuscular mycorrhizal fungus reduced the colonization of the pathogen and the severity of the disease, as determined by reduction in vascular discolouration index. In mycorrhizal plants, the production of phytoalexin compounds was always higher than in the nonmycorrhizal plants. There appeared to be a direct correlation between the concentration of the phytoalexins and the degree of mycorrhizal association. Three different compounds with R f values of 0 23 (I), 0 17 (II) and 0 11 (III) were obtained from mycorrhizal plants. Similar compounds were also found to be induced by an abiotic elicitor CuSO 4. The first compound was identified as an isoflavonoid, daidzein and the other two remain to be identified. These compounds were checked for their antifungal activity in vitro. The germination of conidial spores of Fusarium oxysporum was strongly inhibited by the compound III than the other two. It is argued that the production of phytoalexin compounds in mycorrhizal plant could be one of the mechanisms imparting tolerance of the plants to wilt disease. Keywords. Phytoalexins; mycorrhizae; cowpea; Glomus fasciculatum; disease control. 1. Introduction It has been widely accepted that vesicular-arbuscular mycorrhizal (VAM) fungi enhance plant mineral nutrition especially phosphorus (P) (Mosse 1973; Hayman 1986). Other than their influence on plant nutrition, their interaction with plant pathogens such as fungi, bacteria and nematodes may lead to either the reduction or increase in severity of disease (Schenck et al 1977; Schonbeck 1980; Schenck 1981; Schonbeck and Dehne 1981; Dehne 1982; Bagyaraj 1984). For instance, when tomato plants were inoculated with Glomus mosseae, the damage caused by Fusarium oxysporum f. sp. lycopersici was considerably reduced (Dehne and Schonbeck 1975). Such tolerance, due to mycorrhizal association may be imparted by one or more of the following mechanisms: alterations in the physiology of the host, improvement of the plant nutritional status, anatomical changes and production of phenolic compounds (Baltruschat and Schonbeck 1972; Ling-Lee et al 1977; Dehne et al 1978; Krishna and Bagyaraj 1983; Morandi et al 1984). Of these the production of phytoalexins is considered to be an important mechanism of Corresponding author. 291

2 292 Ρ Sundaresan, Ν Ubalthoose Raja and Ρ Gunasekaran disease resistance in plants (Morandi et al 1984). Only a few reports are available on phytoalexin production in VAM plants. Morandi et al (1984) observed accumulation of isoflavonoid compounds during VAM symbiosis. To understand the role of VAM fungi in disease resistance, we examined the interaction between VAM and a wilt causing fungus, Fusarium oxysporum, in cowpea. Further, the elicitation and accumulation of phytoalexin compounds in response to fungal infection and their antifungal activity were examined in vitro. 2. Materials and methods 2.1 Plant material Seeds of cowpea (Vigna unguiculata (L) Walp.) were obtained from Pulses Research Centre, Pudukkottai, Tamil Nadu. They were planted in a phosphorous-deficient, black sandy loam soil (Sundaresan 1989) containing 4 mg/kg of extractable Ρ (NaHCO 3 -soluble P) and grown in greenhouse condition (approx C, rh 80%, light 16 h, 85 W m-2). 2.2 Fungal cultures An isolate of F. oxysporum used in the present study was originally isolated from diseased cowpea roots and maintained on potato dextrose agar (PDA) at 26 C in dark. A VAM fungus, Glomus fasciculatum (Thaxter sensu Gerd.) Gerd, and Trappe (obtained from Prof. D J Bagyaraj, University of Agricultural Sciences, Bangalore) was maintained in pot cultures with Panicum maximum (Jacq.) (Bagyaraj and Manjunath 1980). 2.3 Preparation of conidial suspension of F. oxysporum F. oxysporum was cultured on PDA for 26 days at 26 C and conidia were collected in sterile Czapek-Dox liquid nutrient containing a trace amount of Tween-80. This suspension was sieved through 45 µm sieve to remove mycelial clumps. The conidial concentration was adjusted to conidia/ml. 2.4 Interaction of VAM with F. oxysporum Three cowpea plants were grown in each pot containing 5 kg of sterilized experimental soil in greenhouse conditions. Prior to sowing, the pots were either inoculated with 500 spores of G. fasciculatum or with 25 ml of conidial suspension of F. oxysporum ( /ml). F. oxysporum was inoculated to both mycorrhizal (MF) and non mycorrhizal (NMF) plants simultaneously or at 10 days intervals. Control, mycorrhizal (Μ) and non-mycorrhizal (NM) plants were also maintained. There were three replicates per treatment and the plants were harvested after 75 days of growth.

3 Phytoalexins in mycorrhizal cowpea Quantification of VAM infection and spore number The VAM colonization was quantified using a modified method of Phillips and Hayman (1970) using 0 5% lactoglycerol-trypan blue after the G. fasciculatum infected roots were cleared in 10% KOH (Kormanik et al 1980). The stained roots were observed under the microscope and the degree of colonization was calculated according to Read et al (1976). The number of VAM fungal spores in the soil was determined after sieving the soil samples (Gerdemann and Nicolson 1963) and observing the soil suspension under the microscope. 2.6 Plant parameters The plants were harvested after 75 days and their weight was determined after drying the samples at 80 C for 24 h. The total Ρ content of the plant was estimated using the method of Allen (1940). 2.7 Calculation of vascular discolouration index Vascular discolouration index (VDI) was calculated as an index of the colonization of F. oxysporum using the following formula (Davis et al 1979). 2.8 Phytoalexin production in cowpea roots Surface sterilized cowpea seeds were germinated in 1 kg of sterile experimental soil. Five seedlings per pot and three replicates for each treatment were maintained. Ten days old cowpea plants were provided with either 50 ml of CuSO 4 (10 3 M) solution (positive control) or with distilled water (negative control). After four days of treatment, the plants were harvested and phytoalexins were extracted in case of abiotic elicitor treated plants. Five hundred spores of G. fasciculatum was given as inoculum to develop mycorrhizal plants and for control plants no inoculum was added. VAM and non-vam plants were harvested at 15, 30, 45 and 60 days intervals. The roots were collected and used for phytoalexin extraction. 2.9 Extraction and characterization of phytoalexins One gram of roots from each treatment was macerated in 10 ml of 95% ethanol. The extract was dried and redissolved in chloroform (2 ml/g fresh weight) and applied to a column (1 10 cm) of silica gel ( mesh, Glaxo, India) equilibrated with chloroform. The column was first eluted with 40 ml of chloroform and later with 20 ml of ethyl acetate : chloroform (1:1 v/v). The fractions were concentrated and applied to a thin-layer chromatography (TLC) plate (0 25 mm *Based on vascular discoloration scale of 0 = No discolouration to 3= 100% discolouration of the xylem tissues in each root.

4 294 Ρ Sundaresan, Ν Ubalthoose Raja and Ρ Gunasekaran thickness) and was developed with a mixture of hexane: diethyl ether (1:3 v/v) Three spots were scraped from the plate and were eluted with 10 ml of ethanol Compounds were further purified by repeated TLC. They were redissolved in ethanol and the maximum absorbance for each compound was measured in spectrophotometer (Hitachi U 2000) Quantitative analysis of phytoalexins Since compound I was identified as daidzein by co-chromatographing with authentic sample daidzein, the extracted phytoalexin was quantified and expressed as µg/g fresh weight of roots. Compounds II and III were not identified and they were quantified with their relative absorbance at their respective absorption maximum Large scale extraction of phytoalexins One kg of VAM roots of cowpea was air dried and repeatedly extracted with hot ethanol. The extracts were combined, made into a slurry by adsorbing it over silica gel ( mesh) and packed in a column (3 40 cm). This was eluted successively with benzene and increasing amounts of acetone. Fractions of 100 ml were collected each time and the solvent was distilled. The residues obtained were subjected to TLC separation using different solvent systems. The residue of fractions 1 to 21 (fraction A; eluted with 5, 10, 15 and 20% of acetone in benzene) had the properties of wax and therefore was discarded. The residues of fractions 22 to 30 (fraction B; eluted with 25 and 30 % of acetone in benzene), fractions 31 to 43 (fraction C; eluted with 35, 40, 45 and 50 % of acetone in benzene) and fractions 44 to 55 (fraction D; eluted with 55 and 60% of acetone in benzene) had a single compound each with minor impurities. However, these three compounds were found to be different from each other (R f values were 0 11, 0 17 and 0 23 for the fractions B, C and D respectively). They were recrystallized from methanol, dissolved in ethanol and their UV spectrum recorded. As compounds obtained from fractions Β and C were in small quantity, the melting points for them could not be determined. However, fraction D yielded sufficient amount to determine the melting point Antifungal activity of phytoalexin compounds in vitro The antifungal activity of the phytoalexins separated from the large scale purification was checked in vitro. Stock solutions (0 2%) of each phytoalexin in ethanol were prepared and 10, 25, 75 and 100 μg were dispersed into multiwell assay trays. The volume of each well was made up to 50 µl by the addition of ethanol and then 950 μl of a conidial suspension containing fungal conidia ( ) in Czapek-Dox liquid nutrient was added to each well. There were three replications for each treatment. Controls, without phytoalexin were kept in all assays. The assay trays were incubated for three days at 26 C in dark. The lowest concentration at which no mycelial development occurred was taken as the minimum inhibitory concentration (MIC).

5 3. Results Phytoalexins in mycorrhizal cowpea Tolerance of mycorrhizal plants to F. oxysporum infection Plants inoculated with G. fasciculatum (M) showed a significant increase in plant biomass, whereas the F. oxysporum inoculated non-mycorrhizal plants (NMF) showed the lowest biomass (3 90 g/plant) and recorded maximum VDI (74 1%) (table 1). However, when F. oxysporum was inoculated simultaneously with VAM, the biomass of the plants was more than the F. oxysporum inoculated plants and the VDI was 51 6%. When F. oxysporum was inoculated after the establishment of VAM fungus in the roots, the VDI was much less. For example, F. oxysporum inoculated after 30 days of VAM establishment showed 14 8% VDI and about 19 2% reduction in the severity of disease. The disease tolerance of the mycorrhizal plants was not correlated to their Ρ status. Mycorrhizal plants showed a marked increase (2 6 times) in Ρ content over the non-vam plants. F. oxysporum treated non-mycorrhizal plants (NMF) showed a significant reduction in the Ρ level. On the contrary, plants simultaneously inoculated with F. oxysporum and G. fasciculatum (MF) showed higher Ρ content. The reduction in Ρ status of mycorrhizal plant was found to be less if F. oxysporum was inoculated to established mycorrhizal plants. Table 1. Interaction between G. fasciculatum and F. oxysporum after inoculating them at various intervals. Values are mean of 3 replicates. Values not followed by the same latter are significantly different (P=0.05, Duncan s new multiple range test). NM and Μ denote non-mycorrhizal and mycorrhizal plants respectively. F10, F20 and F30 denote the day of Fusarium inoculation that followed the VAM inoculation. 3.2 Development of VAM with Fusarium in roots The VAM colonization was considerably reduced when F. oxysporum was inoculated simultaneously with G. fasciculatum. However, this damage caused by the pathogen was reduced when the pathogen was inoculated after the establishment of VAM fungus in roots (table 1). Simultaneous inoculation of both the organisms reduced the VAM spore production in soil. However, when the

6 296 Ρ Sundaresan, Ν Ubalthoose Raja and Ρ Gunasekaran pathogen was inoculated in the later stages, the reduction in spore number was substantial. These results suggested that the VAM development control invasion of the pathogen not merely by increasing the Ρ level of the plant. studied the production of phytoalexin compounds in the VAM plants and relevance to development of resistance of plants to the pathogen. 3.3 Separation and identification of phytoalexins Treatment with both the elicitors increased the accumulation of phytoalexins in roots. From CuSO 4 -treated and VAM colonized roots, three compounds with different R f values (0 23, 0 17 and 0 11) were separated by TLC. The absorbance maxima of these compounds were found to be 248, 283 and 340 nm respectively. The first compound which had a R f value of 0 23 and an absorbance maximum of 248 nm (figure 1) showed a melting point at 319 C and was identified as an isoflavonoid compound, daidzein. Figure 2 shows the absorbtion maxima of compounds II and III extracted from VAM plants. Figure 1. Maximal absorbance of daidzein pure and compound I.

7 Phytoalexins in mycorrhizal cowpea 297 Figure 2. Maximal absorbance of compounds II and III. 3.4 Accumulation of phytoalexins in roots When cowpea plants were treated with CuSO 4, phytoalexin production was enhanced with about 9 7-, and 13-fold increase in the concentration of compounds I, II and III respectively over the untreated plants (table 2). VAM infection in plants greatly induced the synthesis of phytoalexin compounds; the extent of phytoalexin production was positively correlated with the degree of VAM colonization (table 3). At the final harvest, VAM plants had 3 0, 5 0 and 4 0 times more concentration of daidzein, compounds II and III respectively than the nonmycorrhizal plants. Table 2. Synthesis of phytoalexin compounds in cowpea roots elicited with CuSO 4. Values are the means of three replicates. *Expressed in µg/g fresh weight. **The concentration of compounds II and III Were expressed in relative absorbance at 283 and 340 nm respectively since the compound were not identified. Values are the absorbance of compounds separated from 1 g of fresh roots 3.5 Antifungal activity of phytoalexins in vitro Among the three compounds extracted from VAM roots, compound III showed a strong inhibition of fungal growth (table 4). The minimum inhibitory concentration

8 298 Ρ Sundaresan, Ν Ubalthoose Raja and Ρ Gunasekaran Table 3. Phytoalexin accumulation in cowpea roots in relation to the development of VAM infection. *Expressed in µg/g fresh weight **The concentration of compounds II and III were expressed in relative absorbance at 283 and 340 nm respectively since the compounds were not identified. Values are the absorbance of compounds separate from 10 g of fresh roots. Table 4. Inhibitory concentration of phytoalexins on germination of F. oxysporum spores. Values are the means of 3 replicates of this compound for F. oxysporum spore germination was found to be 10 µg/ml, whereas for compound II, it was 50 µg/ml. The antifungal effect was very low with daidzein and the MIC was > 100 µg/ml. 4. Discussion There is increasing evidence on the interaction of VAM fungi and plant pathogenic organisms (Schenck and Kellam 1978; Schonbeck 1980; Schenck 1981; Schonbeck and Dehne 1981). Since VAM are established in the roots of host plants, it can primarily reduce the diseases caused by soil-borne pathogens (Dehne 1982). In the study, the interaction of G. fasciculatum with a wilt causing pathogen F. oxysporum and the production of phytoalexins were investigated. F. oxysporum infection lowered the plant growth and correspondingly increased the vascular discolouration index. However, simultaneous inoculation with VAM fungus G. fasciculatum reduced the severity of disease caused by the pathogen. Similar observations have been reported for other host-vam fungus-pathogen combinations (Baltruschat and Schonbeck 1972; Dehne and Schonbeck 1975; Schenck et al 1975; Davis and Menge 1981; Chakravarty and Mishra 1986). In a few instances VAM fungus inoculation were also reported to increase or have no effect on the development of fungal root pathogen (Ross 1972; Davis et al 1978; Davis 1980). Disease incidence was substantially reduced when pathogens were inoculated to roots already colonized by the VAM fungus. In fact a clear negative association between the extent of VAM

9 Phytoalexins in mycorrhizal cowpea 299 colonization and severity of disease caused by the pathogen has been found. Similar observation with F. oxysporum f. sp. lycopersici (Dehne and Schonbeck 1975) and Phytophthora parasitica (Schenck et al 1977) have been reported. Increased resistance of mycorrhizal plants to disease have been attributed to several mechanisms. One of the possible mechanisms of resistance of VAM plants to the pathogen could be because of the improved mineral nutrition, particularly P. VAM inoculated plants had an increased Ρ level and exhibited decreased VDI. Krishna and Bagyaraj (1983) suggested the possible role of Ρ in disease tolerance in a G. fasciculatum-sclerotium rolfsii-peanut interaction system. Alternatively because mycorrhizal roots are more lignified especially in the stelar tissue they may restrict the entry of pathogen into the root cortex (Dehne 1982). Increased level of phenols especially ortho-dihydroxy phenols in the mycorrhizal plants (Ling-Lee et al 1977; Krishna and Bagyaraj 1984) has also been argued to impart disease resistance (Goodman et al 1967; Krishna and Bagyaraj 1983). Dehne et al (1978) suggested that the production of chitinase like hydrolytic enzymes in mycorrhizal plants may also act on the pathogens. The increased resistance of mycorrhizal roots to pathogen may be due to the alteration of host physiology by the accumulation of specific amino acids such as arginine which was found to decrease the sporulation of Thielaviopsis basicola (Baltruschat and Schonbeck 1972; Schonbeck and Dehne 1977; Dehne et al 1978). Finally, the resistance of mycorrhizal plants could also be due to phytoalexins (Bailey 1982; Mansfield 1982). Our results showed that mycorrhizal infection increased the production of phytoalexins in cowpea. Further, the extent of accumulation of phytoalexin compounds was positively associated with colonization of G. fasciculatum. The production of phytoalexins was also found to be elicited by CuSO 4. Earlier, Morandi et al (1984) reported an increase in the accumulation of phytoalexin Compounds in soybean with increase in infection by G. mosse and G. fasciculatum; they also reported elicitation of phytoalexins by CuSO 4. In our study, three compounds with different absorption maxima were separated and one of them was identified as daidzein. Synthesis of daidzein, an intermediate in the biosynthetic pathway of isoflavonoids such as coumestrol and glyceollin, has also been found in soybean-glomus symbiosis (Morandi et al 1984). Compound III having an absorption maxima at 340 nm strongly inhibited Fusarium spore germination. Daidzein was found to be less active against the pathogen. No report is available on the toxic effect of daidzein on either fungi or nematodes (Morandi et al 1984). Based on our results, we suggest that mycorrhizal association in plants may restrict the development of pathogen by the production of phytoalexins. Thus, inoculation of the VAM fungus. G. fasciculatum, to cowpea not only improves plant growth and development, but offers increasing resistance to soil-borne pathogens such as F. oxysporum. Acknowledgements Authors acknowledge the Indian Council of Agricultural Research, New Delhi, for the financial assistance. Dr Μ Ramaiah and Dr Η R Krishnan, Department of Natural Products, School of Chemistry, Madurai Kamaraj University, Madurai, for their help in the large scale extraction of phytoalexins and Prof. Μ Lakshmanan for his help during the project.

10 300 Ρ Sundaresan, Ν Ubalthoose Raja and Ρ Gunasekaran References Allen R J L 1940 The estimation of phosphorus; Biochem. J. B Bagyaraj D J 1984 Biological interactions with mycorrhizal fungi; in VA mycorrhiza (eds) C L Powell and D J Bagyaraj (New York: CRC Press) pp Bagyaraj D J and Manjunath A 1980 Selection of a suitable host for mass production of VA mycorrhizal inoculum; Plant Soil Bailey J A 1982 Mechanisms of phytoalexin accumulation; in Phytoalexins (eds) J A Bailey and J W Mansfield (Glasgow: Blackie) pp Baltruschat Η and Schonbeck F 1972 The influence of endotrophic mycorrhiza on the infestation of tobacco by Thielaviopsis basicola; Phytopathol. Z Chakravarty Ρ and Mishra R R 1986 Influence of endotrophic mycorrhiza on the Fusarium wilt of Cassia tora L; J. Phytopathol Davis R Μ 1980 Influence of Glomus fasciculatus on Thielaviopsis basicola root rot of citrus; Plant Dis Davis R Μ and Menge J A 1981 Phytophthora parasitica inoculation and intensity of vesiculararbuscular mycorrhiza in citus; New Phytol Davis R M, Menge J A and Erwin D C 1979 Influence of Glomus fasciculatus and soil phosphorus on Verticillium wilt of cotton; Phytopathology Davis R M, Menge J A and Zentmyer G A 1978 Influence of vesicular-arbuscular mycorrhizae on Phytophthora root rot of three crop plants; Phytopathology Dehne Η W 1982 Interaction between vesicular-arbuscular mycorrhizal fungi and plant pathogens; Phytopathology Dehne Η W and Schonbeck F 1975 The influence of the endotrophic mycorrhiza on the fusarial wilt of tomato; Z. Pflanzenkr. Pflanzenschutz Dehne Η W, Schonbeck F and Baltruschat Η 1978 The influence of endotrophic mycorrhiza on plant diseases 3. Chitinase-activity and the omithine-cycle; Z. Pßanzenkr. Pflanzenschutz Gerdemann J W and Nicolson Τ Η 1963 Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting; Trans. Br. Mycol. Soc Goodman R N, Kiraly Ζ and Zaitlin Μ 1967 The biochemistry and physiology of infectious plant disease; (Princeton: Van Nostrand) pp Hayman D S 1986 Mycorrhizae of nitrogen-fixation legumes; MIRCEN J. Appl. Microbiol Biotechnol Kormanik Ρ Ρ, Bryan W C and Schultz R C 1980 Procedures and equipment for staining large numbers of plant root samples for endomycorrhizal assay; Can. J. Microbiol Krishna Κ R and Bagyaraj D J 1983 Interaction between Glomus fasciculatum and Sclerotium rolfsii in peanut; Can. J. Bot Krishna Κ R and Bagyaraj D J 1984 Phenols in mycorrhizal roots of Arachis hypogea; Experientia Ling-Lee M, Chilvers G A and Ashford Α Ε 1977 A histochemical study of phenolic materials in mycorrhizal and uninfected roots of Eucalyptus fastigata Deane and Maiden; New Phytol Mansfield J W 1982 The role of phytoalexins in disease resistance; in Phytoalexins (eds). J A Bailey and J W Mansfield (Glasgow: Blackie) pp Morandi D, Bailey J A and Gianinazzi-Person V 1984 Isoflavonoid accumulation in soybean roots infected with vesicular-arbuscular mycorrhizal fungi; Physiol. Plant Pathol Mosse Β 1973 Advances in the study of vesicular-arbuscular mycorrhiza; Annu. Rev. Phytopathol Phillips J Μ and Hayman D S 1970 Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection; Trans. Br. Mycol. Soc Read D J, Koucheki Η Κ and Hodson J 1976 Vesicular-arbuscular mycorrhiza in natural vegetation system, I The occurrence of infection; New Phytol Ross J Ρ 1972 Influence of Endogone mycorrhiza on phytophthora rot of soybean; Phytopathology Schenck Ν C 1981 Can mycorrhiza control root disease?; Plant Dis Schenck Ν C and Kellam Μ Κ 1978 The influence of vesicular-arbuscular mycorrhizae on disease development; Fα. Agric. Exp. Stn. Bull 799

11 Phytoalexins in mycorrhizal cowpea 301 Schenck Ν C, Kinloch R A and Dickson D W 1975 Interaction of endomycorrhizal fungi and root knot nematode on soybean; in Endomycorrhizas (eds) F Ε Sanders, Β Mosse and Ρ Β Tinker (London: Academic Press) pp Schenck Ν C, Ridings W Η and Cornell J A 1977 Interaction of two vesicular-arbuscular mycorrhizal fungi and Phytophthora parasitica of two citrus root stocks, in Proceedings of the third North American Conference on Mycorrhizae (Oregon: Corvallis) p 9 Schonbeck F 1980 Endomycorrhiza: Ecology, function and phytopathological aspects; Forum Microbiol Schonbeck F and Dehne Η W 1977 Damage to mycorrhizal and non-mycorrhizal cotton seedlings by Thielaviopsis basicola; Plant Dis. Rep Schonbeck F and Dehne Η W 1981 Mycorrhiza and plant health; Gesunde Pflanz Sundaresan Ρ 1989 The factors influencing the symbiosis between host plant Vigna unguiculata and the mycorrhizal fungus Glomus fasciculatum, Ph.D. thesis, Madurai Kamaraj University, Madurai.